牛传染性鼻气管炎病毒gE蛋白的真核表达及其多克隆抗体的制备

Eukaryotic expression of gE protein of infectious bovine rhinotracheitis virus and preparation of polyclonal antibody

目的真核表达及纯化牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)gE蛋白,并制备其多克隆抗体。方法合成IBRV gE基因全长,并加入KpnⅠ和BSTBⅠ酶切位点,连接至真核表达载体pCDNA4-MycHis,构建重组质粒pCDNA4-gE-His,经双酶切鉴定正确后转染MDBK细胞系,镍柱纯化带有His标签的重组gE蛋白。将纯化后的重组gE蛋白皮下多点注射新西兰白兔,制备多克隆抗体并纯化,利用Dot blot和间接ELISA方法分析其抗原性。结果重组质粒pCDNA4-gE-His经双酶切鉴定证明构建正确,转染MDBK细胞系后,表达的重组gE蛋白相对分子质量约为61000,纯化后浓度为50μg/mL。制备的多克隆抗体纯化后浓度为1 mg/mL,经Dot blot和ELISA方法鉴定,重组gE蛋白具有良好的抗原性。结论真核表达了IBRV重组gE蛋白,成功制备了抗gE多克隆抗体,表达产物具有良好的抗原性,为诊断方法的建立以及疫苗和IBRV致病机制等相关研究奠定基础。

Objective To express the g E protein of infectious bovine rhinotracheitis virus(IBRV)in eukaryotic cells,purify the expressed product and prepare its polyclonal antibody.Methods Full-length gE gene of IBRV was synthesized,to which the restriction sites of KpnⅠand BSTBⅠwere introduced,and inserted into eukaryotic expression vector pCDNA4-Myc-His.The constructed recombinant plasmid pCDNA4-gE-His was identified by restriction analysis and transfected to MDBK cells.His-tagged recombinant gE protein was purified by Ni-NTA Agarous Kit,with which rabbits were immunized by subcutaneous injection in several sites.The prepared polyclonal antibody against gE was purified and analyzed for antigenicity by Dot blot and indirect ELISA.Results Restriction analysis and sequencing proved that recombinant plasmid pCDNA4-gE-His was constructed correctly.Recombinant gE protein with a relative molecular mass of 61000 was expressed in MDBK cells transfected with the recombinant plasmid,and reached a concentration of 50μg/mL after purification.The prepared polyclonal antibodies against gE protein reached a concentration of 1 mg/mL after purification,and showed good antigenicity as proved by Dot blot and ELISA.Conclusion The IBRV gE protein was expressed in MDBK cells,against which the polyclonal antibody was successfully prepared and showed good antigenicity.It laid a foundation of development of diagnostic method and vaccine as well as study on pathogenic mechanism of IBRV.